Cloning Pp523, a downy mildew resistance gene of Brassica oleracea L.

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On the Way to the Identification of a Downy Mildew Resistance Gene in Brassica oleracea L.

Publication . Carlier, Jorge; Alabaca, C; Rodrigues, C.; Leitão, José

We have recently mapped the dominant and monogenically inherited downy mildew resistance locus Pp523 to the chromosome C8 of Brassica oleracea L. and constructed a physical-map of the genomic region of this locus. Presently, we are constructing a BAC genomic library of a downy mildew resistant S4 line derived from the broccoli accession originally found to harbor the Pp523 locus and using STS markers derived from the previously constructed physical map in order to identify a contig of BAC clones which sequencing could allow the isolation and cloning of this resistance locus. The new BAC library contains 17280 clones with an average insert size of 125 Kbp, which represent approximately 3.5 equivalents of the broccoli genome. To date, 18 clones from this new library were selected and assembled in a contig (with two small gaps) that covers the major part of the previously physically mapped region. The overlapping between BAC clones has been identified by PCR and confirmed by sequencing of the PCR products. The precise positioning of BAC clones in the genome has been made possible through the alignment of the BAC-end sequences with the genomic sequences of B. oleracea available at the Brassica database (BRAD).

2013Conference object

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Physical mapping in a triplicated genome: mapping the downy mildew resistance locus Pp523 in Brassica oleracea L.

Publication . Carlier, Jorge; Alabaça, Cláudia S.; Sousa, N. H.; Coelho, P. S.; Monteiro, A. A.; Paterson, A. H.; Leitão, J. M.

We describe the construction of a BAC contig and identification of a minimal tiling path that encompass the dominant and monogenically inherited downy mildew resistance locus Pp523 of Brassica oleracea L. The selection of BAC clones for construction of the physical map was carried out by screening gridded BAC libraries with DNA overgo probes derived from both genetically mapped DNA markers flanking the locus of interest and BAC-end sequences that align to Arabidopsis thaliana sequences within the previously identified syntenic region. The selected BAC clones consistently mapped to three different genomic regions of B. oleracea. Although 83 BAC clones were accurately mapped within a similar to 4.6 cM region surrounding the downy mildew resistance locus Pp523, a subset of 33 BAC clones mapped to another region on chromosome C8 that was similar to 60 cM away from the resistance gene, and a subset of 63 BAC clones mapped to chromosome C5. These results reflect the triplication of the Brassica genomes since their divergence from a common ancestor shared with A. thaliana, and they are consonant with recent analyses of the C genome of Brassica napus. The assembly of a minimal tiling path constituted by 13 (BoT01) BAC clones that span the Pp523 locus sets the stage for map-based cloning of this resistance gene.

2011Journal article

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